Really high enrichment... but equally high IgG? - How is this possible? (Dec/15/2008 )
Hi folks,
i've really hit a wall here. My ChIP is with mouse cells, using nuclease digestion instead of sonication to break up the chromatin. The ChIPs were working well until one day, I started getting super high IgG background signals.
Now, I've been getting 70-90% of input (I use equal volumes of chromatin for quantifying input and for doing IP, so this is effectively a concentrating factor or IP efficiency), which in the first place seems way too high to be real. But I'm getting an equally high value of enrichment for my IgG control (I used to get only 5-10% for IgG)! My PCR replicates look really good, and pos/neg controls also normal, so I don't think it's my PCR. I've tried using more stringent post-IP washing protocols, as well as different IgG antibodys from different companies etc, so it doesn't seem like it's the IgG...
The only suspect I can think of is the DNA extraction. I use this Chelex extraction method to extract my DNA from the magnetic beads which improves PCR results a lot, but I only started having this IgG background problem after I started using this method... even though the first time I used it it was working fabulously...
Also, FYI, my ChIP is using 2000 to 10,000 cells per IP.
Any ideas?
i've really hit a wall here. My ChIP is with mouse cells, using nuclease digestion instead of sonication to break up the chromatin. The ChIPs were working well until one day, I started getting super high IgG background signals.
Now, I've been getting 70-90% of input (I use equal volumes of chromatin for quantifying input and for doing IP, so this is effectively a concentrating factor or IP efficiency), which in the first place seems way too high to be real. But I'm getting an equally high value of enrichment for my IgG control (I used to get only 5-10% for IgG)! My PCR replicates look really good, and pos/neg controls also normal, so I don't think it's my PCR. I've tried using more stringent post-IP washing protocols, as well as different IgG antibodys from different companies etc, so it doesn't seem like it's the IgG...
The only suspect I can think of is the DNA extraction. I use this Chelex extraction method to extract my DNA from the magnetic beads which improves PCR results a lot, but I only started having this IgG background problem after I started using this method... even though the first time I used it it was working fabulously...
Also, FYI, my ChIP is using 2000 to 10,000 cells per IP.
Any ideas?
Sounds like you're just able to get more usable DNA using the chelex. Is the DNA yield better now that you use the chelex method (i.e. lower Cts for qPCR or lower number of cycles to obtain a band with semi-quantitative). When we first started using Fast ChIP we saw a big improvement in the % of input. Since there's really no true extraction you really don't lose any DNA like you would doing phenol:chloroform extractions and precipitations (you even lose some when doing column clean-ups). Also, the high pH of the chelex beads really protects the DNA during any heating steps so I'm guessing that improves the yield.
If basically all that happened was that you're getting a higher DNA yield with the new method then the way to fix the background problem is use less chromatin in your IPs. This can dramatically increase your signal:noise ratio/dynamic range of the assay. Also, if you are not blocking your beads with both protein (BSA) and nucleic acid (sheared salmon sperm DNA, tRNA, etc.) this can help with the background problem as well, though if you are using the magnetic beads I doubt it will help much.